The long term objective of this project is to investigate how microRNAs (miRNAs) tilt the interaction of herpes simplex virus (HSV) and neurons either towards lytic infection or towards latency. HSV latency is the most fascinating biological property ofthe virus and its most important clinical feature. Understanding HSV latency may lead to new therapies or even a cure for this widespread pathogen. MiRNAs are important regulatory molecules in many biological systems, but much remains unknown about how they impact HSV infections. The first specific aim of this project is to determine the roles of host and viral miRNAs in repressing lytic gene expression during latency. Mice with the miRNA processing enzyme Dicer knocked out in a subset of sensory neurons, and viral mutants with lesions that affect specific miRNAs or target sites on specific mRNAs will be used to test the effects of miRNAs on gene expression during establishment and maintenance of latency in vivo and, with Project 3, in vitro. Effects of these mutations on chromatin status will be examined with Project 1. Targets for an interesting miRNA, miR-H5, will be identified using multiple approaches. Oligonucleotides that antagonize miRNA function (anti-mlRs) will be tested for effects on gene expression and reactivation in vitro with Project 3, and, if results are promising, in vivo. The second specific aim is to investigate the roles of HSV-1 miRNAs that counteract innate immunity using viral mutants affected for these miRNAs, with emphasis on whether these miRNAs promote latency by abetting host survival in the face of HSV-1 infection. Effects of these miRNAs in neurons on innate immunity and autophagy will be explored in vitro with Project 3. The third specific aim focuses on the lytic side of the lytic/latent balance, and seeks to determine how HSV-1 miRNAs promote lytic replication and reactivation. How ATRX, a target of HSV-1 miR-H1, is downregulated in lytic infection and how that impacts virus production, chromatin status, and reactivation from latency will be explored with Project 1 in cell culture and in vivo and, with Project 3, in an in vitro latency system, using virus mutants and anti-mlRs. Additional targets for miR-H1 and targets for a conserved family of miRNAs expressed during lytic infection will be identified. RELEVANCE (See instructions); This project proposes research to understand how miRNAs contribute to herpes simplex virus (HSV) latency, which is the most fascinating biological and most vexing clinical aspect of this virus. Understanding HSV latency might lead to new treatments or even cures for HSV infections, which are widespread and, in some cases, life-threatening.